bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.04.04.716481

Determination of suitable reference genes for RT-qPCR analysis in Gryllodes sigillatus (Orthoptera: Gryllidae)

Houda, H. B. M.·Bonhomme, R.·Renois, F.·Deschamps, M.-H.·Benoit-Biancamano, M.-O.·Meurens, F.
View original source ↗Export metadata ↓

Abstract

The tropical house cricket Gryllodes sigillatus is a major species used in the edible insect farming industry. Despite the rapid expansion of this sector, diagnostic tools for detecting infections in these species remain limited. The lack of validated reference genes compromises the reliability of RT-qPCR-based gene expression analyses, which are essential for the development of molecular tools for disease diagnosis and health monitoring in insect production systems. To address this gap, we evaluated the expression stability of six candidate reference genes (ACTB, EF1, GAPDH, HisH3, RPL5, and 18SrRNA) across four body parts (abdomen, head, legs, and whole body) using a combination of complementary statistical approaches, including geNorm, NormFinder, BestKeeper, the {Delta}Ct method, the R statistical environment, and the integrated RefFinder tool. Candidate genes were identified and annotated using the recently published G. sigillatus genome, through sequence comparisons with closely related insect species using BLAST and reciprocal BLAST analyses, multiple sequence alignments. All procedures complied with MIQE 2.0 guidelines to ensure methodological rigor and transparency. The results showed that ACTB, EF1, RPL5, and 18SrRNA exhibited stable and consistent expression across all analyzed tissues, whereas GAPDH and HisH3 displayed high variability and were generally unsuitable for normalization, except in head tissue where GAPDH remained stable. This study provides the first validated set of reference genes for G. sigillatus, establishing a robust foundation for accurate, reproducible, and comparable gene expression analyses. Furthermore, these findings support the development of RT-qPCR-based diagnostic tools, contributing to improved health monitoring and biosafety in insect production systems.

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Houda, H. B. M., Bonhomme, R., Renois, F., Deschamps, M.-H., Benoit-Biancamano, M.-O., Meurens, F.. 2026-04-05. Determination of suitable reference genes for RT-qPCR analysis in Gryllodes sigillatus (Orthoptera: Gryllidae). https://doi.org/10.64898/2026.04.04.716481

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

An ssRNA Phage Single-gene Lysis Protein Targeting MraY Reveals Convergent Evolution Across the RNA-DNA Phage Boundary

Small lytic phages of the ssRNA Leviviricetes and ssDNA Microviricetes achieve host lysis by expression of a single protein, designated as an Sgl protein. Four ssRNA Sgls have been shown to inhibit steps in peptidoglycan biosynthesis: three target MurJ, the Lipid II flippase, and one targets MurA, the committed step of the pathway. A fifth Sgl, the E protein of the canonical ssDNA microvirus {varphi}X174, inhibits MraY, which catalyzes the synthesis of the first lipid-linked intermediate. This inhibition requires SlyD, a cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase. The Sgl of the Caulobacter ssRNA phage {varphi}Cb5 (Cebevirus halophobicum) has not been characterized. Here, we present physiological evidence that Sgl{varphi}Cb5 targets MraY. Moreover, we show that, despite the absence of detectable sequence similarity, Sgl{varphi}Cb5 shares several features with the {varphi}X174 E protein, including the distribution of hydrophobic and charged residues, a proposed N-out, C-in membrane topology, and retention of lytic activity in an Sgl{varphi}Cb5 -LacZ fusion. Furthermore, expression of the Caulobacter cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase CC2799 enhances Sgl{varphi}Cb5 -mediated lysis in E. coli. These parallels between Sgl function in a DNA phage and an RNA phage either represent remarkably consonant convergent evolution or some evolutionary cross-over event for which there is currently no precedent.

molecular biology↗
bioRxiv2026

HERC1-mediated orphan subunit quality control maintains proteasome homeostasis and is disrupted in HERC1-associated neurodevelopmental disease

Biallelic hypomorphic variants in the E3 ubiquitin ligase HERC1 cause a neurological disorder clinically characterized by macrocephaly, dysmorphic facies, and psychomotor delay. Identifying the substrates of HERC1 is essential for understanding its biological functions and the mechanisms underlying disease-associated variants. To address this, we performed a quantitative proteomic analysis using biotinylated ubiquitin to identify HERC1 targets and examine their regulation in cells expressing HERC1 with or without ubiquitin ligase activity. This approach revealed a broad set of HERC1-dependent ubiquitylation substrates, including proteins from major multiprotein complexes such as the proteasome, the translation initiation machinery, and pathways involved in cell metabolism. Focusing on the proteasome, the most prominently represented complex, we identified several regulatory particle subunits, including PSMC6, PSME1, and PSME2. Specifically, within the 19S regulatory particle, we demonstrated that ubiquitylation of the PSMC6 subunit involves the formation of K48-linked polyubiquitin chains and depends on HERC1 activity. Other 19S subunits, such as PSMC2, PSMC4, and PSMC5, undergo a similar modification. Mechanistically, we show that HERC1 selectively recognizes unassembled subunits, promotes their ubiquitylation, and targets them for proteasomal degradation. Notably, in HERC1-deficient cells, disruption of this mechanism impaired proteasomal activity. At the disease level, quantitative proteomic analysis of the pathogenic HERC1 variant c.1448G>A (p.Gly483Glu) likewise identified altered ubiquitylation of proteasome subunits and components of the translation initiation machinery. Biochemical analyses further revealed impaired ubiquitylation of PSMC6 by the mutant HERC1 protein, associated with a loss of interaction between HERC1 and PSMC6. Together, these findings establish a link between HERC1-associated neurological disorders and defective ubiquitylation pathways. They elucidate the molecular mechanisms by which HERC1 regulates proteostasis and demonstrate how disruption of these processes contributes to disease pathogenesis, thereby highlighting potential avenues for therapeutic intervention.

molecular biology↗
bioRxiv2026

PKR inhibition prevents maladaptive responses to endoplasmic reticulum (ER) stress in early-onset dystonia DYT-PRKRA.

DYT-PRKRA is a rare, childhood-onset dystonia caused by mutations in the PRKRA gene, which encodes the protein PACT. PACT activates protein kinase R (PKR) in response to endoplasmic reticulum (ER) stress. In DYT-PRKRA patient cells, the PACT-PKR interaction is enhanced, increasing sensitivity to ER stress-induced apoptosis. The canonical protein kinase activated by ER stress is PKR-like endoplasmic reticulum kinase (PERK), which phosphorylates the subunit of eukaryotic translation initiation factor 2 (eIF2) to initiate the integrated stress response (ISR) and restore cellular homeostasis via the transcription factor ATF4. However, under persistent stress, ATF4 induces CHOP, shifting the adaptive response toward apoptosis. PKR, also activated by ER stress, phosphorylates eIF2, but its contribution to cellular homeostasis remains understudied. This work shows, for the first time, that PKR activation is essential for CHOP induction during ISR, directly linking enhanced apoptosis in DYT-PRKRA cells to prolonged PKR activation and heightened CHOP induction. Strikingly, a small-molecule PKR inhibitor effectively blocks CHOP expression and significantly decreases apoptosis in DYT-PRKRA cells, suggesting PKR inhibition as an effective therapeutic strategy for DYT-PRKRA.

molecular biology↗